Liver transplantation remains the only effective therapy for end-stage liver failure but is limited by a severe shortage of donor livers. Moreover, initial poor function and primary nonfunction frequently occur, especially in recipients of fatty liver grafts. Still unknown are changes that occur during cold ischemic storage that predispose to graft injury after transplantation. During the previous period of support, we developed the hypothesis that ATP depletion during cold storage inhibits the proton-pumping vacuolar ATPase (V-ATPase) in lysosomes and collapses lysosomal acidic pH gradients, which causes release of lysosomal Fe2+ into the cytosol through divalent metal transporter-1 (DMT1). Mitochondria take up this Fe2+ via the mitochondrial Ca2+ (and Fe2+) uni-porter (MCU) to predispose to Fe2+-dependent hydroxyl radical formation in mitochondria after transplantation with consequent activation of c-Jun N-terminal kinase (JNK), onset of the mitochondrial permeability transition (MPT) and cell death. Pursuing this hypothesis, we propose five specific aims. In Aim 1, intracellular iron mobilization in hepatocytes of cold-stored mouse livers will be measured by multiphoton microscopy of calcein, an iron-indicating fluorophore. We will assess the lysosomal origin of iron and the role of MCU using the lysosomally targeted iron chelator, starch-desferal, and the MCU inhibitors, minocycline and doxycycline. Further, we will assess protection by iron chelators and MCU inhibitors against graft injury and failure after rat liver trans- plantation, including assessment of mitochondrial function after transplantation by innovative intravital mul- tiphoton microscopic techniques. In Aim 2, a similar analysis will be performed for Kupffer cells (KCs) and sinusoidal endothelial cells (SECs) to test the hypothesis that lysosomal iron mobilization also promotes KC activation and loss of SEC viability, which are prominent early features of hepatic storage/reperfusion injury. In Aim 3, the role of DMT1 in lysosomal iron release during liver storage and graft injury after transplantation will be assessed using pharmacological inhibitors. Using novel transgenic mice created with cre-lox technology, the effect of cell-specific DMT1 deficiency in hepatocytes, KCs and SECs on iron release and graft injury will be assessed. These experiments will identify which cell types are most important to iron translocation- dependent graft dysfunction. For Aim 4, we hypothesize that lysosomal iron release during cold storage promotes JNK activation after reperfusion. Using iron chelators and MCU inhibitors, we will determine whether iron translocation to cytosol or mitochondria is most important for JNK activation and the relative importance of different cell types to iron-dependent JNK activation. Lastly in Aim 5, we will characterize the importance of lysosomal iron mobilization to enhanced storage/reperfusion injury in steatotic livers induced by a high-fat, high-fructose diet. We expect earlier iron mobilization by fatty livers during cold storage that promotes greater graft dysfunction and failure after transplantation. Success of this project will lead to novel translatable strategies and new drug targets against storage/reperfusion injury.